Abstract Two new drimenyl cyclohexenone derivatives, named purpurogemutantin 1 and purpurogemutantidin 2, and the known macrophorin A 3 were isolated from a bioactive mutant BD-1-6 obtained by random diethyl sulfate DES mutagenesis of a marine-derived Penicillium purpurogenum G59. Structures and absolute configurations of 1 and 2 were determined by extensive spectroscopic methods, especially 2D NMR and electronic circular dichroism ECD analysis. Possible biosynthetic pathways for 1–3 were also proposed and discussed. Compounds 1 and 2 significantly inhibited human cancer K562, HL-60, HeLa, BGC-823 and MCF-7 cells, and compound 3 also inhibited the K562 and HL-60 cells. Both bioassay and chemical analysis HPLC, LC-ESIMS demonstrated that the parent strain G59 did not produce 1–3, and that DES-induced mutations in the mutant BD-1-6 activated some silent biosynthetic pathways in the parent strain G59, including one set for 1–3 production. View Full-Text